Fresenius' Zeitschrift für Analytische Chemie .Eine Methode zur zuverlässigen Produktion hochempfindlicher

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  • Fresenius' Zeitschrift für

    Analytische Chemie Labor- und Betriebsverfahren

    Herausgegeben von W.Fresenius

    unter Mitwirkung der Fachgruppe Analytische Chemie der Gesellschaft Deutscher Chemiker

    vertreten durch H.Bode H.Kienitz G.Tölg H.Specker

    International Editorial Board D. Betteridge J. T. Clerc A. Dijkstra H.Malissa E.Pungor Yu.A.Zolotov

    Band 290 · 1978

    Springer-Verlag Berlin • Heidelberg • New York J. F. Bergmann München

  • The exclusive copyright for all languages and countries, including the right for photomechanical and any other reproductions, also in microform, is transferred to the publisher. The use in this journal of registered or trade names, trademarks etc. without special acknowledgement does not imply that such names, as defined by the relevant protection laws, may be regarded as unprotected and thus free for general use.

    Alle Rechte, einschließlich das der Übersetzung in fremde Sprachen und das der fotomechanischen Wiedergabe, auch in Mikroform, oder einer sonstigen Verviel­ fältigung, vorbehalten. Jedoch wird gewerblichen Unternehmen für den innerbetrieb­ lichen Gebrauch nach Maßgabe des zwischen dem Börsenverein des Deutschen Buchhandels e.V. und dem Bundesverband der Deutschen Industrie abgeschlosse­ nen Rahmenabkommens die Anfertigung einer fotomechanischen Vervielfältigung gestattet. Wenn für diese Zeitschrift kein Pauschalabkommen mit dem Verlag ver­ einbart worden ist, ist eine Wertmarke im Betrage von DM 0,40 pro Seite zu ver­ wenden. Der Verlag läßt diese Beträge den Autorenverbänden zufließen.

    Die Wiedergabe von Gebrauchsnamen, Handelsnamen, Warenbezeichnungen usw. in dieser Zeitschrift berechtigt auch ohne besondere Kennzeichnung nicht zu der Annahme, daß solche Namen im Sinne der Warenzeichen- und Markenschutz- Gesetzgebung als frei zu betrachten wären und daher von jedermann benutzt werden dürften.

    Springer-Verlag · Berlin · Heidelberg · New York

    Printed in Germany by Wiesbadener Graphische Betriebe GmbH, Wiesbaden

    © by Springer-Verlag Berlin · Heidelberg 1978

  • I I. R a d i o i m m u n o a s s a y 11 163

    11.2.

    Fresenius Ζ. Anal . Chem. 290, 163 (1978) - < by Springer-Verlag 1978

    A Method for the Consistent Production of High Quality Antisera to Small Peptide Hormones*

    M . V. Sofronicw, M . Madlcr. O. A . Müller, and P. C. Scriba

    Anatomische Anstalt und Medizinische Klinik Innenstadt, Universität München, Pcltcnkofcrstr. 11. D-8000 München 2

    Eine Methode zur zuverlässigen Produktion hochempfindlicher Antiseren gegen Peptidhormone

    Key words: Radioimmunoassay; Herstellung von Antiseren gegen Peptidhormone

    Specific, high affinity and high titer antisera arc needed for radioimmunological or immunohistochemical methods, as well as for in vivo physiological studies, often requiring large amounts of antiserum. The difficulties in producing high quality antisera against small peptides, including A C T H and arginine-vasoprcssin (AVP) , are well known. Using the high antigenicity of thyroglobulin (Tg) as a carrier protein [2] and the intradermal injection technique [3] in a regimen of repeated injections of small amounts of antigen, we have been successful in consistently producing high quality antiscra against these two peptides in a large number of animals.

    Methods. The peptide was covalenti) bound to bovine Tg using water soluble l-ethyl-3(3-dimethylaminopropyi)-car- bodiimide (CD1) in molar ratios of 60:1:112 (peptide: Tg: GDI), for each immunization of each group of 6 rabbits the following amount of conjugate was prepared: 1 5 mg of Tg plus 1 mg of synthetic A V P (Sehwarz-Mann or Ferring) or 2.4 mg of synthetic A C T H ( / M - 2 3 Corlicotropin-amide, Hoechst) were dissolved in 450 μΙ of water; 150μ1 of freshly prepared CDI-solution (23 mg, 5 ml Water) were added and incubated for 18 h at room tempera­ ture. Then. 600 μ 1 of 1.0 M hydroxylamine were added and incubated for 5 h at room temperature. The resulting con­ jugate was emulsified in 2 ml of Freund's adjuvant, and was divided among the 6 rabbits. For the first 8 injections complete Freund's adjuvant was used, thereafter incomplete. Intradermal injections were made in as many sites as possible. Accompanying the first injection. BCG-vaccine and Pertussis-vaccine were given to each rabbit. Pertussis-vaccine was also given for the 2nd —4th injections. Injections were given at 2 week intervals, with 3 months pauses between the 4th and 5th. and the 8th and 9th. After the 10th injection.

    * Supported bv the Deutsche Forschungsgemeinschaft (grant We 608 3 and SFB 51)

    animals were boostered as often as desired (every 4 — 8 weeks). Animals were bled regularly through the lateral ear veins, between 8 and 12days after all injections.

    Results. The amount of peptide effectively bound to the Tg ranged from 15 — 40 ( , ( ( . tested by conjugation with radio- labelled peptides.

    AVP. Two groups of 6 rabbits were immunized. A l l rabbits surviving after the 4th injection (n — 12) produced antisera. Seven rabbits produced antisera with titers over 1: 100 000. Tiler is defined as end-dilution of antiserum in assav volume (0.5 ml for A V P and 0.7ml for A C T H ) at which 50"„ of labelled peptide is precipitated. The best titers were around 1 : 800 000 (// - 2). Three of these antisera yielded standard curves consistently sensitive below 1 pg of A V P per tube. Plasma determinations with one of these antisera have been published [1].

    ACTH. Two groups of 6 rabbits were immunized. A l l rabbits surviving after the 4th injection (// = 9) produced antisera. One animal died soon after. Four of the eight surviving rabbits produced antisera with titers over 1:100 000. The titers of the antisera of the other four animals ranged from 1 : 14 000 to 1 : 56 000. The best titer was around 17220 000. A l l eight surviving rabbits yielded antisera with standard curves consistently sensitive below 5 pg A C T H per tube. Three rabbits yielded curves consistently sensitive below 2 pg A C T H per tube.

    Using this method, high quality antisera have also been raised to G n - R H . oxytocin and secretin. The A VP- . A C T H - , oxytocin-, and Gn-RH-antiscra have also been successfully used in immunohistochemical methods [4]. Animals immu­ nized against A V P developed a strong diabetes insipidus, those against A C T H had adrenal atrophy, and those against G n - R N gonadal atrophy.

    Conclusion. The high success ratio of this method for production of high quality antisera against several small peptides, suggests that this method may be useful for other small antigens. Successful preliminary results with Prostaglandine F and thyroxine support this assumption.

    References

    1. Hayward. J. N . . Pavasulhipaisit. K . . Perez-Lopez. F. R.. Sofronicw. M . V . : Fndocrinolouv 98. 975 (1976)

    2. Skowskv. W. R.. Fisher. D. Α.: Τ Lab. Cl in . Med. 80. 134 (1972)

    3. Vaitukaitis. J., Robbins. J. B.. Nieschlag, F.. Ross. G . T.: J. Clin, Fndocrin. Metab. 33. 988 (197Ï)

    4. Weindl, Α.. Sofronicw. M . V . : In: R. Fahlbusch. Κ. ν. Werder (Fds.): Pituitary Adenomas. Diagnosis and Treatment. Stuttgart: Thieme 1978

  • Inhalt /Contents

    Band 290 Heft 1 1978 erschienen am/published 20. III. 1978

    Originalabhandlungen

    N. G. McTaggart, L. A. Luke 1 Molekularsiebe für die Erdölanalyse (in Englisch)

    K. Korn 10 Bestimmung von Metallgehalten im Sickerwasser aus Mülldeponien durch Atomabsorptionsspektroskopie

    F. Drawert, H. Krempl, W. Osterrieder, S. Sipos Die voltammetrische Reaktionsführung der Reaktion zwischen Phenolen und N.N-Diäthyl-p-Phenylendiamin

    A. Zeman, P. Bartl, A. Schaaff, V. Christ 21 Analyse synthetischer Flugturbinenöle durch Gas-Chromato- graphie-Massenspektrometrie. Teil I. Pentaerythritesteröle

    B. Mann, Ο. Schrems 29 Eine quantitative Anwendung der Ferninfrarot-Fourier- Spektroskopie (in Englisch)

    W. G. Albrecht 33 Abtrennung durch Diasolyse mit Hilfe gequollener Silicongummimembranen. 2. Mitteilung

    D. Saur 40 E m elektromechanischer Tropfkontroller für die Quecksilber- tropfelektrode

    D. Saur 42 E m Digitalsweepgenerator für die Polarographie

    Original Papers

    N. G. McTaggart, L. A. Luke Molecular Sieves for the Analysis of Petroleum

    K. Korn Determination of Metals in Leachate by Atomic Absorption Spectrophotometry (in German)

    15 F. Drawert, H. Krempl, W. Osterrieder, S. Sipos Voltammetric Conduction of the Reaction between Phenols and Ν,Ν-Diethyl-p-phenylenediamine (in German)

    A. Zeman, P. Bartl, A. Schaaff, V. Christ Analysis of Synthetic Aviation Turbine Oils by Gas Chromatography—Mass Spectrometry. Part I. Pentaerythritol Ester Oils (in German)

    B. Mann, O. Schrems A Quantitative Application of Far-Infrared Fourier Spectroscopy

    W. G. Albrecht Separation by Diasolysis by means of Swollen Silicone Rubber Membranes. 2. Communication (in German)

    D. Saur An Electromechanical Drop-Controller for the Dropping Mercury Electrode (in German)

    D. Saur A Digital Sweep Generator for Polarography (in German)

    Kurze Mitteilungen

    J. G. Schindler, G. Stork, H.-J. Strüh, W. Schäl 45 Siloxanverbindungen als aktive Komponenten in ionen­ selektiven Elektrodenmembranen

    S. P. Srivastava, V. K. Dua, K. Gupta 46 Dünnschicht-chromatographische Trennung einiger anorganischer Ionen (in Englisch)

    R. Kuroda, N. Yoshikuni, K. Tateno 46 Trenn